Enzyme-loaded pollen-mimicking microparticles for organophosphate detoxification of insect pollinators

ABSTRACT

A composition for detoxifying insect pollinators from one or more organophosphate pesticides, the composition containing microparticles comprising: (i) a phosphotriesterase; (ii) nanoparticles; and (iii) a surface active agent. Also disclosed herein is an aqueous suspension comprising the above-described detoxifying microparticles in an external aqueous medium, which may also contain an insect pollinator attractant. Also described herein is a method for detoxifying insect pollinators of one or more organophosphate pesticides, the method comprising placing the detoxifying aqueous suspension in a location accessible to the insect pollinators to permit the insect pollinators to ingest the detoxifying aqueous suspension, wherein the detoxifying aqueous suspension comprises microparticles, as described above, suspended in an external aqueous medium, typically also including an insect pollinator attractant in the external aqueous medium.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 63/122,692, filed on Dec. 8, 2020, which is hereinincorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant number2017-18-107 awarded by the National Institute of Food and Agriculture,US Department of Agriculture, Hatch, and under Award NumberR21-NS10383-01 awarded by the Counter ACT Program of the NationalInstitute of Health, and under Award Number IIP-1918981 awarded by theNational Science Foundation. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention generally relates to methods for detoxifying orpreventing toxification of insect pollinators, such as bees. The presentinvention more particularly relates to detoxifying or preventingtoxification of insect pollinators by feeding the insect pollinators acomposition that degrades or removes a toxifying compound, such as apesticide toxic to the insect pollinators.

BACKGROUND OF THE INVENTION

One-third of U.S. crops are dependent on managed and native bees forsustained production, yield, and quality. Pollinators contribute $24billion to the U.S. economy, of which honeybees are responsible for over$15 billion (The White House Archives, 2014, Fact Sheet: The EconomicChallenge Posed By Declining Pollinator Populations, Washington D.C.:Office of the Press Secretary). However, bee populations are rapidlydeclining (Kulhanek, K., et al., Journal Of Apicultural Research, 56(4),328-340, 2017. https://doi.org/10.1080/00218839.2017.1344496).Beekeepers lose on average one-third of colonies each winter and morethan 700 U.S. native bee species are now at risk of extinction (TheCenter for Biological Diversity, 2017. Pollinators In Peril. PortlandOreg.). Between 2013-2019, the U.S. beekeeping industry spent $2billion, a third of its income, on replacing 10 million hives (Amadeo,K. (2019). Bee Colony Collapse Disorder and Its Impact on the Economy.The Balance). The loss of managed colonies has caused a rise inpollination fees for many crop farmers. Growers are currently facingdiminished crop yields as a result of poor pollination from weakenedcolonies.

The application of insecticides, and other agrochemicals is consideredto be a major cause of managed and native bee population loss (D.Goulson et al., Science, 347, 10, 2015). Insecticides can have lethaland sub-lethal effects on pollinators, both at an individual and colonylevel, often through the impairment of vital neuronal pathways (J. Yaoet al., Journal of Economic Entomology, 111, 4, August 2018, 1517-1525).Other agrochemicals, such as fungicides, can cause synergistic effectswith other toxins by destroying beneficial gut bacteria, which areessential for defending against viruses, parasites and insecticides (A.Iverson et al., Apidologie, 50, 733-744, 2019). In the case of socialbees, chemicals can be transported back into the hive within pollen andnectar and accumulate within wax, developing brood and other bee castes(S. McArt et al., Sci. Rep., 7, 46554, 2017). Research has shown overthe course of a year up to 93 different foreign compounds accumulatedwithin a colony and that the number of pesticides was a strong predictorof colony death (K. Traynor et al., Sci. Rep., 6, 33207, 2016).

Organophosphate (OP) pesticides, in particular, are heavily relied uponin agricultural production to prevent crop loss due to numerous types ofinsects. OPs have a market of over $7 billion and account for more thana third of insecticide sales worldwide and often lead to pollinatorexposures and exhibit high toxicity towards honey bees and bumble bees(S. R. Rissato et al., Food Chem., 101, 1719-1726, 2007). OPinsecticides influence insect cholinergic neural signaling throughinhibition of carboxyl ester hydrolases, particularlyacetylcholinesterase (AChE) which breaks down acetylcholine. OPsinactivate AChE through irreversible covalent inhibition, causing abuild-up of acetylcholine and overstimulation of nicotinic andmuscarinic receptors (J. V. Peter et al., Indian J. Crit. Care Med., 18,735, 2014).

Some examples of organophosphate pesticides include malathion,parathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos,phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, azinphos-methyl,azinphos-ethyl, and terbufos. Malathion and parathion are the two of themost widely applied OPs in commercially pollinated crops. Malaoxon,malathion's metabolite, is 1000-fold stronger at inhibiting AChE thanmalathion (0. P. Rodriguez et al., Bull. Environ. Contam. Toxicol., 58,171-176, 1997). Malathion and parathion exhibit oral LD₅₀'s of 0.38 and0.175 μg/bee respectively (C. D. S. Tomlin, The Insecticide Manual: AWorld Compendium, British Crop Production Council, ISBN 9781901396188,2009).

Although these pesticides are useful in mitigating the damage caused byagricultural pests, they unfortunately also have an adverse effect oninsect pollinators, such as bees (e.g., honey bees and bumble bees), asdiscussed above. As insect pollinators are critical for agriculture andfarming, the use of such pesticides can result in a critical decline inpollination, which represents a threat to global food production andecological balance.

SUMMARY OF THE INVENTION

The present invention provides a downstream solution to the persistentand pernicious problem of inadvertent pesticide toxification of insectpollinators, particularly bees. To achieve the solution, a method isherein described for detoxifying insect pollinators that have ingestedone or more organophosphate pesticides. The method more particularlyinvolves feeding a detoxifying formulation to a community of insectpollinators, wherein the detoxifying formulation includes microparticlescontaining: i) a phosphotriesterase; (ii) nanoparticles; and (iii) asurface active agent. The phosphotriesterase functions to hydrolyze theorganophosphate pesticide inside the insect pollinator when ingested bythe insect pollinator. The nanoparticles may have an inorganiccomposition (e.g., a carbonate) or organic composition (e.g., abiodegradable polyester). In some embodiments, the phosphotriesterase isdissolved or suspended in an aqueous medium (i.e., internal aqueousmedium) contained within each microparticle.

Typically, the microparticles are provided to the insect pollinators inthe form of a suspension of the microparticles in an external aqueousmedium, typically with an insect pollinator attractant included in theexternal aqueous medium. The method is typically practiced by placingthe detoxifying composition, typically as an aqueous suspension, in alocation accessible to the insect pollinators to permit the insectpollinators to ingest the detoxifying composition.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. A schematic of the passage of microparticles through a beedigestive tract. Microparticles analogous to pollen grains move into themidgut as they are extracted by the proventriculus, which drawsparticulates out of the crop stomach. The PIM structure protects theencapsulated protein from gastric acidity. PIMs are retained in themidgut to detoxify pesticides as they are released during pollendigestion.

FIGS. 2A-2G. Characterizations and analysis of the stability, sizedistribution and morphology of PIMs. FIG. 2A (panels left, middle, andright) provides microscopic images of unmodified CaCO₃ microparticles(control) in pH 7.4 (left panel) and PIMs in pH 7.4 (middle panel) andpH of 4.8 (right panel). Note: CMP denotes unmodified CaCO₃microparticles, included as the control. Insets are highermagnifications. FIG. 2B plots size distribution of PIMs and unmodifiedmicroparticles in pH 7.4 and PIMs in pH 7.4 and 4.8. FIG. 2C plotsrelative suspension stability of unmodified microparticles and PIMs in 2g ml⁻¹ sucrose. FIG. 2D shows sucrose solution (left panel) and largescale PIM suspension (right panel). FIG. 2E provides morphologicalanalysis and size distribution analysis of PIMs fabricated at a largescale. FIG. 2F (panels left, middle, and right) shows SEM images ofmicroparticles at different magnifications (as indicated) to determinePIM surface morphology. FIG. 2G shows pore size distribution analysis ofPIMs and PIMs loaded with HSA. The data are presented as means, anderror bars represent the standard deviation.

FIGS. 3A-3G. Characterizations of OPT encapsulation and activity inPIMs. FIG. 3A shows fluorescent imaging of PIMs containingCy5.5-modified gelatin (middle panel), FITC-conjugated HAS (left panel),and a merge image (right panel). FIG. 3B shows protein loadingefficiency (PLE) of CMP and PIM loaded with HSA at 5, 10 and 15% PFC.FIG. 3C shows PLE of PIM loaded with OPT at 2% and 5% PFC. FIG. 3D showsrelative activity of OPT-PIM and free OPT in paraoxon hydrolysis underpH 7.4 and 4.8 (n=3). FIG. 3E shows relative activity of OPT-PIM andfree OPT in malathion hydrolysis under pH 7.4 and 4.8 (n=3). FIG. 3Fshows temperature-dependent relative activity of OPT-PIM and free OPT inparaoxon hydrolysis when incubated at temperatures 30, 40, 50 and 60° C.(n=3). FIG. 3G shows long-term relative activity of OPT-PIM and free OPTin paraoxon hydrolysis when stored at room temperature and 4° C. (n=3).Statistical analysis was performed by using one-way ANOVA tests (FIGS.3D and 3E) and two-way ANOVA tests (FIGS. 3F and 3G). Data are presentedas means and error bars represent the standard deviation. NS, nostatistical significance; room temperature (r.t.).

FIGS. 4A-4B. Tracking of digested PIMs by fluorescent imaging ofbumblebee GI tracts. FIG. 4A shows GI tracts following HSA-PIMtreatment; fluorescence was maintained up to 12 h post-consumption.Microparticle morphology was clearly visible and microparticles weresuccessfully drawn into the midgut (n=3; relatively brighter backgroundat 1 and 12 h was probably due to protein leakage during digestion).FIG. 4B shows GI tracts following free-HSA treatment (n=3).

FIGS. 5A-5F. Characterization of OPT-PIM efficacy through AChE activityassay and bee survival experiments. FIG. 5A is a schematic showing thatformation of thiocholine from acetylthiocholine through AChE cleavagecan be characterized using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB).DTNB and thiocholine react to form TNB²⁻, the absorbance of which can bemeasured at 412 nm. FIG. 5B shows relative activity of AChE fromhomogenized honeybees when incubated in 0.5 mM paraoxon or DI water (thepositive control) and treated with samples of free OPT, OPT-PIM and DIwater (the negative control). For this experiment, n=9. The positivecontrol is homogenized honeybee cells without any paraoxon treatment;the negative control is homogenized honeybee cells treated with paraoxonbut no free OPT or OPT-PIMs treatment. FIG. 5C depicts an exemplaryapparatus for determining mortality following contaminated pollen ballconsumption against PIM treatment in syrup. FIG. 5D shows the survivalrate of bumblebees following acute exposure to paraoxon (50 μg g⁻¹pollen) over 12 hours when treated with 500 μg ml⁻¹ OPT treatments(n=40). FIG. 5E is a plot of exposure to paraoxon (15 μg g⁻¹ pollen)over 10 days (n=50). FIG. 5F is a plot of exposure to malathion (750 μgg⁻¹ pollen) over 10 days (n=50). Statistical analysis was performed byusing one-way ANOVA tests (FIGS. 5B and 5D). Data are presented as meansand error bars represent the standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present disclosure is directed to a composition fordetoxifying insect pollinators that have ingested or otherwiseinternalized one or more organophosphate (OP) compounds or substances,typically used as pesticides. The term “pesticide,” as used herein,broadly includes any substance applied onto plants to improve thequality, growth, or product yield of the plants. The pesticide generallypossesses one or more properties of controlling or regulatingagricultural or horticultural pests, wherein the pests may becrop-damaging insects, animals, fungi, or undesired plant life (e.g.,invasive species or weeds). As noted earlier above, although pesticidesare used for controlling or killing crop-damaging insects, agriculturalpesticides are generally not intended for controlling or killing insectpollinators. The pesticide may be, more specifically, an insecticide,herbicide, fungicide, or nematicide. For purposes of the presentdisclosure, the pesticide is an organophosphate compound or substance.Some examples of organophosphate pesticides include malathion,parathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos,phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, azinphos-methyl,azinphos-ethyl, and terbufos.

A first component (component i) of the detoxifying composition is aphosphotriesterase. The phosphotriesterase, also known as anaryldialkylphosphatase or organophosphate hydrolase, may be any of thetypes (variants or strains) known and may be derived from any bacterialsource. Phosphotriesterases are metalloenzymes that hydrolyze thetriester linkage found in OP insecticides (A. B. Pinjari et al., Lett.Appl. Microbiol., 57, 63-68, 2013). There are several variants ofphosphotriesterase; the most frequently used, amidohydrolasephosphotriesterase (OPT), is isolated from bacteria P. diminuta orFlavobacterium ATCC 27551 and exhibits a TIM-barrel fold structure (Y.Zheng et al., Appl. Biochem. Biotechnol., 136, 233-241, 2007). OPT canbe produced from transfected E. coli culture with the appropriate OPTplasmid sequence (C. Roodveldt et al., Protein Eng. Des. Sel., 18,51-58, 2005). OPT has a wide substrate specificity; it exhibits optimalhydrolysis upon encountering paraoxon (parathion's metabolite), at arate approaching the limit of diffusion (S. R. Caldwell et al.,Biochemistry, 30, 7438-7444, 1991). OPT performs best hydrolyzingsubstrates which possess phenol leaving groups, yet it will alsosuccessfully degrade thiol linkages as in the case of malathion (S. B.Hong et al., Biochemistry, 35, 10904-10912, 1996). Notably, OPTapplication has demonstrated poor efficacy in industry due to its poorstability at a low pH and high temperatures (C. Y. Yang et al.,ChemBioChem, 15, 1761-1764, 2014). Bioactivity rapidly declines at pHsless than 8.0. At pH of 7.0, activity is less than half of its maximumpotential. At the optimum pH range of 8.0-9.5, the Co²⁺ OPT complexmaintains thermostability at less than 45° C., above which, thestability rapidly declines until deactivation at 60° C. (D. Rochu etal., Biochem. J. 380, 627-633, 2004).

A second component (component ii) of the detoxifying composition isnanoparticles. The nanoparticles can have any solid composition,provided that the solid composition is non-toxic to insect pollinators.The nanoparticles should also have a composition that is substantiallyinsoluble in water or aqueous solution and also non-reactive with water.The nanoparticles can have a particle size of, for example, 1, 2, 5, 10,20, 30, 40, 50, 100, 200, 250, 300, 350, 400, 450, or 500 nm, or aparticle size within a range bounded by any two of the foregoing values,e.g., 1-500 nm, 1-200 nm, 1-100 nm, or 1-50 nm. In some embodiments, anyrange of nanoparticle sizes derivable from the above values may beexcluded.

In a first set of embodiments, the nanoparticles have an inorganiccomposition. The inorganic composition may be a non-toxic salt, such asa carbonate or sulfate salt, or combination thereof. Some examples ofcarbonate salts include sodium carbonate, potassium carbonate, calciumcarbonate, and magnesium carbonate. Some examples of sulfate saltsinclude sodium sulfate, potassium sulfate, calcium sulfate, andmagnesium sulfate. The carbonate composition may or may not contain aportion thereof in sulfate form, typically no more than 20 wt % of thecarbonate composition. The inorganic composition may alternatively be ametal oxide or metal sulfide composition, wherein the “metal” refers toany element substantially non-toxic to insect pollinators. The metal maybe, for example, an alkaline earth element, transition metal element, ormain group element. Some examples of metal oxide compositions includesilicon oxide (silica), aluminum oxide (alumina), zinc oxide, magnesiumoxide, calcium oxide, titanium oxide, yttrium oxide (yttria), andzirconium oxide. Analogous metal sulfide compositions can be derived byreplacing oxide with sulfide in any of the foregoing examples.

In a second set of embodiments, the nanoparticles have an organiccomposition. The organic composition may be a polymer composition. Someexamples of organic polymeric compositions include polyethylene,polypropylene, polyethylene terephthalate, polyurethanes, polyamides,polycarbonates, polyureas, vinyl addition polymers (e.g., polyacrylate,polymethylacrylate, polymethacrylate, polymethylmethacrylate,polyvinylalcohol, and polyvinylacetate) and biodegradable esterpolymeric compositions, such as polylactic acid (PLA) and polyglycolicacid (PGA).

In some embodiments, the nanoparticles may have an acid-scavengingcomposition, with carbonate compositions being exemplary. Theacid-scavenging composition may be inorganic or organic and should becapable of neutralizing an acid and/or maintaining an alkaline conditionin an aqueous medium in contact with the acid-scavenging composition.

A third component (component iii) of the detoxifying composition is asurface active agent. The surface active agent should be non-toxic toinsect pollinators. The surface active agent may be any substance knownin the art to have a surface active property, i.e., surfactant ability,including any of the non-toxic surfactants known in the art. The surfaceactive agent may be, for example, a natural or synthetic polymer. Insome embodiments, the surface active agent is a natural-basedsurfactant, such as a polypeptide (e.g., protein) or polysaccharide(sugar or carbohydrate). Some examples of polypeptide surface activeagents include gelatin, collagen, fibrin, polylysine, and polyaspartate.Some examples of polysaccharide surface active agents include dextran,dextrose, starch, maltodextrin, chitosan, pectin, agarose, hemicellulose(e.g., xylan), alginate, carrageenan, guar gum, xanthan gum, locust beangum, and cellulose gum. The surface active agent may alternatively beamphiphilic by containing one or more hydrophilic portions and one ormore hydrophobic sections. Some examples of amphiphilic surface activeagents include sodium lauryl sulfate, alkylbenzene sulfonates, andlignin sulfonates. Some examples of synthetic polymers include polyvinylalcohol, polyvinyl acetate, and polysorbate-type non-ionic surfactants(e.g., polysorbate 80).

The surface active agent may alternatively be a non-ionic surfactant,which typically contains at least one polyalkylene oxide (hydrophilic)portion attached to a hydrophobic hydrocarbon portion. The polyalkyleneoxide (PAO) portion is typically polyethylene oxide (PEO), althoughpolypropylene oxide (PPO), and polybutylene oxide (PBO) may also serveas the PAO. The PAO typically includes at least or greater than 5, 10,15, 20, 30, 40, or 50 alkylene oxide units. As part of the hydrophilicportion, the non-ionic surfactant may alternatively or in additioninclude one or more hydroxy (OH) or cyclic ether (e.g., tetrahydrofuran)groups per molecule. The hydrocarbon portion is generally constructedsolely of carbon and hydrogen atoms, except that one or more fluorineatoms may or may not be present. The hydrocarbon portion may be orinclude one or more alkyl groups, alkenyl groups, cycloalkyl groups, andaromatic groups (e.g., phenyl). In some embodiments, the non-ionicsurfactant includes a hydrocarbon group corresponding to a linear orbranched hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl group.Some examples of non-ionic surfactants include: (i) Triton® X-100 andIgepal® surfactants, which contain a (1,1,3,3-tetramethylbutyl)phenylportion; (ii) polysorbate (Tween®) surfactants, such as polysorbate 80,which contain a polyethoxylated sorbitan moiety attached (typically viaan ester bond) to a hydrocarbon group, such as an undecyl group; (iii)non-ionic triblock copolymers, also known as poloxamers, such asPluronic® surfactants, which typically contain alternating PEO and PPOunits, such as PEO—PPO-PEO and PPO-PEO-PPO surfactants; and (iv) Brij®surfactants, which contain a PEO portion attached to an alkyl portion(typically 12-20 carbon atoms).

The above described components (i)-(iii) are included as components ofmicroparticles. The end result is that the microparticles are composedof at least or solely components (i)-(iii). In some embodiments, thephosphotriesterase and surface active agent are dispersed throughouteach microparticle. In other embodiments, the phosphotriesterase iscontained within a core portion of the microparticle, with thenanoparticles forming a shell surrounding (encapsulating) thephosphotriesterase core. The surface active agent may be in the core,shell, or both. The encapsulation of the phosphotriesterase provides theadvantage of protecting the phosphotriesterase from unfavorable acidicGI conditions within the insect pollinator. In further embodiments, thephosphotriesterase may be dissolved or suspended in an aqueous mediumwithin each microparticle. The aqueous medium can have any suitable pH,particularly an alkaline pH, such as a pH of at least or greater than 7,7.5, 8, 8.5, 9, 9.5, or 10, or a pH within a range bounded by any two ofthe foregoing values.

The microparticles may or may not include one or more additionalcomponents. In one embodiment, the microparticles further include aninsect pollinator attractant admixed with components (i), (ii), and/or(iii). In another embodiment, the microparticles further include pollenadmixed with components (i), (ii), and/or (iii). In another embodiment,the microparticles further include one or more nutrients for insectpollinators. The one or more nutrients may be, for example, one or morecarbohydrates (e.g., sugar or nectar), amino acids, vitamins, minerals,or lipids (e.g., fatty acids or sterols).

The microparticles typically have a size of at least 0.1 microns and upto 200 microns. In different embodiments, the microparticles have a sizeof precisely, about, at least, up to, or less than 0.1, 0.2, 0.5, 1, 2,5, 10, 20, 30, 40, 50, 100, 150, or 200 microns, or a size within arange bounded by any two of the foregoing values (e.g., 0.1-200 microns,0.1-150 microns, 0.1-100 microns, 0.1-50 microns, 1-200 microns, 1-150microns, 1-100 microns, 1-50 microns, 10-200 microns, 10-150 microns,10-100 microns, or 10-50 microns). In some embodiments, any range ofmicroparticle sizes derivable from the above values may be excluded.

The microparticles may also possess an outer surface porosity, with thepores typically being nanosized, such as 1-500 nm or 1-100 nm in size.Typically, the pores correspond to interstitial spaces between thenanoparticles. In different embodiments, the pores have a size ofprecisely, about, at least, greater than, up to, or less than, forexample, 1, 2, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or500 nm, or a pore size within a range bounded by any two of theforegoing values.

In another aspect, the present disclosure is directed to a detoxifyingaqueous suspension containing any of the detoxifying microparticlesdescribed above suspended in an external aqueous medium. The externalaqueous medium can have any suitable pH, particularly an alkaline pH,such as a pH of at least or greater than 7, 7.5, 8, 8.5, 9, 9.5, or 10,or a pH within a range bounded by any two of the foregoing values. Atleast when being used to administer to insect pollinators, thedetoxifying aqueous suspension typically contains an insect pollinatorattractant in the external aqueous medium, the detoxifyingmicroparticles, or both. The insect pollinator attractant may be orinclude, for example, sucrose, a plant extract, fruit extract, or apheromone. The attractant may be present in an amount of, for example,1-5 g/mL in the external aqueous medium. However, in some embodiments,an attractant is not included. In some embodiments, the external aqueousmedium includes a surface active agent to help stabilize the suspension.The external aqueous medium may also include one or more auxiliaryagents, such as, for example, a buffer, anti-bacterial agent, ornutrient appropriate for insect pollinators. In some embodiments, thesuspended microparticles are mixed with pollen to form a macroscopicpollen ball, which is then administered to the insect pollinators in thesame manner described above, such as in the form of an aqueoussuspension.

In another aspect, the present disclosure is directed to a method forusing the detoxifying composition to protect insect pollinators from theharmful effects of organophosphate pesticides. The insect pollinatorstypically belong to the order Hymenoptera, such as bees (e.g., honeybees or bumble bees) or wasps. In the method, the detoxifyingcomposition in the form of microparticles or suspension thereof, asdescribed above, is placed in a location accessible to the insectpollinators to permit the insect pollinators to ingest the detoxifyingcomposition. Upon ingestion, the phosphotriesterase functions tohydrolyze the organophosphate pesticide inside the insect pollinator. Insome embodiments, the method results in at least or above 50%, 60%, 70%,80%, or 90% survival of the insect pollinators compared to insectpollinators administered an external aqueous medium without thedetoxifying microparticles.

Typically, the microparticles are provided to the insect pollinators inthe form of a suspension of the microparticles in an external aqueousmedium, as described above, typically with an insect pollinatorattractant included in the external aqueous medium. The attractant maybe present in the external aqueous medium in an amount of, for example,1-5 g/mL in the external aqueous medium. The insect pollinatorattractant may be or include, for example, sucrose, a plant extract,fruit extract, or a pheromone. The method is typically practiced byplacing the detoxifying aqueous suspension in a location accessible tothe insect pollinators to permit the insect pollinators to ingest thedetoxifying aqueous suspension.

Examples have been set forth below for the purpose of illustration andto describe the best mode of the invention at the present time. However,the scope of this invention is not to be in any way limited by theexamples set forth herein.

Examples

Overview

The following experiments describe a low-cost, scalable in vivodetoxification strategy for removing organophosphate insecticides frominsect pollinators. The method involves encapsulation ofphosphotriesterase (OPT) in pollen-inspired microparticles (PIMs).Uniform and consumable PIMs were developed with capability of loadingOPT at 90% efficiency and protecting OPT from degradation in the pH of abee gut. Microcolonies of Bombus impatiens fed malathion-contaminatedpollen patties demonstrated 100% survival when fed OPT-PIMs but 0%survival with OPT alone, or with plain sucrose within five and fourdays, respectively. Thus, the detrimental effects of malathion wereeliminated when bees consumed OPT-PIMs. This design presents a versatiletreatment that can be integrated into supplemental feeds such as pollenpatties or dietary syrup for managed pollinators to reduce risk oforganophosphate insecticides.

Herein is reported a biomaterial approach to control organophosphatetoxicity aimed at managed bees (that is, bumblebees such as the commoneastern bumblebee, Bombus impatiens, or the western honeybee, Apismellifera) using OPT-loaded microparticles (FIG. 1). B. impatiens wasused for the in vivo assays, although a similar gut pH exists for A.mellifera; thus, the results may be relevant to A. mellifera as well.Calcium carbonate microparticles were chosen to deliver OPT on the basisof several design considerations.

First, the microparticles mimic pollen grains in size and are thereforeeasily consumed by bees. Both bumblebees and honeybees have agastrointestinal (GI) tract composed of a crop and ventriculus separatedby a proventricular valve which mechanistically extracts micro-sizedparticles for digestion.

Second, by harnessing the acid scavenging capability of CaCO₃, themicroparticles can protect OPT from unfavorable acidic GI conditions tomaintain enzyme bioactivity once they are consumed by bees. The pH ofthe crop and ventriculus are 4.8 and 6.5, respectively, well below theoptimal pH conditions of OPT.

Third, CaCO₃ microparticles (2-50 μm) are relatively easy andinexpensive to produce in large quantities and are capable of loadingbiomacromolecules during production. With optimized fabricationparameters and, importantly, the inclusion of gelatin as an additive,homogenously sized microparticles were produced that encapsulated OPT at˜90% efficiency and displayed a superior suspension stability insucrose. In vitro studies confirmed the protective effect of themicroparticles on OPT bioactivity. The OPT-encapsulated pollen-inspiredmicroparticles (OPT-PIMs) allowed 100% survival of microcolonies of beesfed malathion-contaminated pollen patties, while 0% survival wasobserved for those fed with OPT alone or plain sucrose after 5 and 4days, respectively.

To understand the protective properties and stability of PIMs, bees werefed PIMs loaded with a FITC-labelled protein, human serum albumin(HSA-PIMs). Fluorescent imaging confirmed almost complete extraction ofPIMs out of the crop stomach by 1 hour and their stability throughoutdigestion for 12 hours. This versatile, scalable, low-costdetoxification strategy can act as a precautionary or remedial measurefor managed pollinators when pollinating in areas of organophosphateapplication, to address the issue of pollinator exposures.

Methods

OPT synthesis. Ampicillin, chloramphenicol and IPTG solutions weresterilized before use. E. coli bearing pQE30-PTE was cultured in Millergrade LB broth containing 100 μg ml-1 ampicillin and 25 μg ml⁻¹chloramphenicol at 37° C. Once cultures in 5,000 ml flasks reachedoptical density (OD) 0.4, 500 μl CoCl₂ (1 M) was added, and at OD0.8-1.0, 500 μl IPTG (200 mg ml-1) was added for every liter of culture.The culture was left for a further 3 hours before collecting. Theculture was then centrifuged for 10 min at 1,333×g in 11 centrifugetubes, the supernatant was removed and the cell pellet was resuspendedin 40 ml resuspension buffer (3.15 g Tris-HCl, 29.22 g NaCl, 56 gglycerol, 44 μl CoCl₂ (1 M), 144 mg imidazole, 1 l H₂O). The solutionwas then sonicated at 65% amplitude (5 s on, 25 s off) for 20 min in anice bath. The solution was subsequently centrifuged for 1.5 hours at4,333×g and the supernatant collected as crude OPT. Crude OPT waspurified using a histidine-select NTA-nickel bead affinity column. Thecolumn was equilibrated using an equilibration buffer (20 mM phosphatebuffer, 300 mM NaCl, 10 mM imidazole) before crude OPT was run throughthe column and washed with further equilibration buffer. Captured OPTwas then eluted with elution buffer (20 mM phosphate buffer, 300 mMNaCl, 250 mM imidazole). OPT was concentrated using Amicon Ultra 15 ml3-kDa-membrane tubes and washed with saline three times. OPTconcentration was determined using a BCA protein assay kit. Confirmationof OPT production was confirmed using SDS-PAGE.

OPT-PIM fabrication. In a 10 ml vial, 1 ml of each of the following wasadded in order and mixed continuously for 10 s using a magnetic stirrerat 6,000 r.p.m.: 24 mg ml⁻¹ gelatin from porcine skin, OPT 3.364 mg ml⁻¹(5% PFC) or OPT 1.345 mg ml⁻¹ (2% PFC), 0.33 M CaCl₂) and 0.33 M Na₂CO₃,to form OPT-PIMs. The solution was centrifuged at 1,000×g for 3 min andthe supernatant subsequently removed. The remaining microparticles weresuspended in either distilled water or 2 g ml⁻¹ sucrose to form 0.5 mgml-1 OPT. The experiment was carried out ten times through theseexperiments.

Microparticle morphology. Microparticle morphology was analyzed byresuspending PIMs in 1 ml of distilled H₂O following centrifugation andanalyzing a drop of the solution under an EVOS FL microscope. To produceCaCO₃ microparticles to compare as a standard, the microparticlefabrication process was repeated without gelatin; distilled H₂O wasadded in substitute. Lyophilized microparticles were furthered analyzedunder SEM.

Results and Discussion

Characterizations of PIMs. Calcium carbonate microparticles can beeasily fabricated by rapidly mixing equimolar 0.33 M solutions of CaCl₂and Na₂CO₃. Size and shape can be acutely controlled by alteringsynthesis parameters such as stirring speed, time and additiveinclusion. Initially, CaCO₃ microparticles were fabricated with noinclusion of an additive, nor control of stirring time. The productdisplayed high incidences of aggregation, calcite crystal growth andpoor size homogeneity (FIG. 2A); the average diameter was around 8.2±5.7μm with a large size distribution under pH 7.4, which caused poorsuspension stability.

To circumvent these challenges, stirring time was restricted to 10 s andincluded gelatin (24 mg ml⁻¹) as an additive, which resulted in smallerand consistently homogenously sized (3.9±0.7 μm) microparticles. Gelatinwas chosen because it is an easily obtained, low-cost natural additive.It is known that the zeta potential of gelatin is −13.2 mV (ref. 42.)and it could thus interact with Ca²⁺ to form a gelatin-Ca complex thatacts as a nucleation agent, subsequently enhancing microparticlestability. Given that these microparticles can be digested by bees insimilar ways to pollen grains, the microparticles are herein alsoreferred to as pollen-inspired microparticles or PIMs (FIG. 2B). PIMsdisplayed a superior suspension stability in sucrose.

The significantly improved suspension stability was confirmed using abiophotometer that measured the uppermost layer of the microparticlesuspension. After 2 days, ca. 90% of unmodified microparticles hadsettled while >75% of PIMs maintained good suspension stability. PIMstook 6 days to fully settle, whereas unmodified microparticles only took3 d (FIG. 2C). The sucrose media used to suspend the microparticles wasat a typical concentration used to feed wintering honeybees (2 g ml⁻¹).Although the molecular mechanism behind crystal growth and aggregationis unclear, scanning electron microscope (SEM) imaging confirmed thatthe gelatin-modified microparticles maintained a highly porousnanoparticle aggregation structure (FIG. 2F). Nanometer-size pores canprovide accessible channels for biomacromolecule diffusion and a highinternal surface area to allow physical adsorption with high substrateloading.

Since OPT-PIMs need to maintain function when passing through aciditypresented by the crop stomach, the PIM stability was tested in pH 4.8for 30 min PIMs at pH 4.8 displayed a fractional shift to a smaller sizedistribution (3.4±0.6 μm) (FIG. 2B). When the test was extended to 1.5h, the PIMs still largely retained their shape, although the averageparticle size further decreased to 1.4±0.4 μm. The PIM fabricationprocess was then repeated using high reagent volumes to demonstrate thecapacity for large-scale manufacture. PIMs were successfully produced ata 1 L total volume (FIG. 2D) and displayed a size distributioncomparable to that of PIMs fabricated at small scale, with an averagesize of 4.3±1.4 μm (FIG. 2E). Microparticle pore size was analyzed inaccordance with density functional theory using N2 adsorption isotherms.PIM nanochannel volumes dropped from 0.0067 to 0.0043 cm³ g⁻¹ followingHSA encapsulation, which further confirmed protein loading (FIG. 2G).Nanochannel diameters only dropped from 14 to 12 nm, which indicatedthat protein loading did not block channels and would still permit OPsto enter for enzymatic degradation.

In vitro degradation of organophosphate pesticides with OPT-PIMs.Protein loading and gelatin modification of PIMs was further confirmedthrough confocal laser scanning microscopy. HSA was used in thisinstance as a model protein. Microparticles exhibited an overlay ofCy5.5-conjugated gelatin and FITC-conjugated HAS (FITC-HSA) in the fullmorphology of each microparticle (FIG. 3A). The confocal laser scanningmicroscopy (CLSM) imaging indicated gelatin conjugation and proteinloading throughout the microparticle volume. The protein loadingefficiency (PLE, percentage of protein loaded inside the microparticlesrelative to the total amount of protein added), as characterizedspectrophotometrically using the FITC-HSA, varied as a function of theprotein feeding content (PFC, percentage of the total amount of proteinadded relative to the total mass of the protein and microparticles).From the PLE and PFC, the protein loading capacity (PLC, the totalentrapped amount of protein divided by the total mass of theprotein-loaded microparticles) was also calculated. HSA-PIMs presented ahigh PLE of 85.5% (PLC=4.31%) for gelatin-modified PIMs and 83.6%(PLC=4.21%) for unmodified microparticles at 5% PFC (FIG. 3B). Theloading efficiency decreased as the PFC increased. For PIMs, a PLE of67.1% (PLC=6.94%) was obtained at 10% PFC, and a PLE of 52.1%(PLC=8.42%) was obtained at 15% PFC. In the case of unmodifiedmicroparticles, a PLE of 64.1% (PLC=6.65%) and a PLE of 47.1%(PLC=7.67%) were obtained, respectively (FIG. 3B).

Considering the loading efficiency decrease at higher PFCs and theintrinsic value of OPT, 5% was selected as a baseline to test OPTloading in PIMs. OPT presented 88.1% PLE (PLC=4.43%) at 5% PFC and 90.1%PLE (PLC=1.81%) at 2% PFC (FIG. 3C). It was herein found that aconcentration of 0.5 mg ml⁻¹ OPT was sufficient to initiate rapidhydrolysis of methyl paraoxon to visibly form nitrophenol. A 2% PFCyielded an OPT concentration of 1.21 mg ml⁻¹ in OPT-PIMs, which could befurther diluted to 0.5 mg ml⁻¹; this dilution offered adequate sucroseto render the solution sufficiently attractive to bumblebees forconsumption. Furthermore, it was herein found that no protein wasreleased from the PIMs up to 7 d following fabrication while suspendedin 2 g ml⁻¹ sucrose.

It is known that OPT catalytic efficiency and conformational stabilitycan vary on structural mutagenesis and variation in the central metalcation. In the present experiments, wild-type Co²⁺-boundphosphotriesterase (molecular weight 39 kDa) was used, which is theoptimum metalloenzyme complex capable of a K_(cat) K_(m) ⁻¹ of 7.6×10⁷M⁻¹ s⁻¹ in hydrolyzing paraoxon, where K_(cat) is the turnover numberand describes how many substrate molecules are transformed into productsper unit time by a single enzyme, and K_(m) gives a description of theaffinity of the substrate to the active site of the enzyme.

For the successful function of the design, it is critical that OPT-PIMsare able to maintain bioactivity in the conditions of a bee digestivetract (pH 4.8 in the crop stomach). Therefore, bioactivity and enzymestability of OPT-PIMs and free OPT were assessed in vitro over a pHrange using OPT 0.5 mg ml⁻¹ and either 0.5 mM paraoxon or 0.44 mMmalathion. Paraoxon assays were carried out by measuring the absorbanceof nitrophenol as it is produced from the OPT-catalyzed degradation ofparaoxon. The relative enzymatic activity was obtained by normalizingthe absorbances of both free OPT and OPT-PIMs to that of OPT-PIMsincubated at pH 7.4. As shown in FIG. 3D, free OPT yielded an activityof 49.7%, approximately half that of OPT-PIMs at pH 7.4. Meanwhile, theK_(m) was determined to be 1.83 mM for OPT-PIMs, lower than that of freeOPT (4.80 mM) in 2 g ml⁻¹ sucrose. The maximum velocity (Vmax) ofOPT-PIMs (0.45 mM min⁻¹) was approximately three times that of free OPT(0.13 mM min⁻¹). Without being bound by theory, it is possible that thehigher performance of OPT-PIMs occurs because microparticleencapsulation facilitates enhanced enzyme kinetics. In addition, theCaCO₃ element of the microparticle structure should possess an ‘acidscavenging’ ability, which could neutralize acid in the microparticle'simmediate vicinity, thus allowing encapsulated OPT to outperform freeenzyme in acidic conditions. OPT-PIMs at pH 4.8 displayed lower activity(73.4%) than that at pH 7.4. However, free enzyme assays almost did notfunction at all (1.7%) at pH 4.8.

An absorbance from malathion hydrolysis was characterized using Ellman'sreagent (5,5′-dithiobis-2-nitrobenzoic acid or DTNB), which can reactwith the thiol group of malathion's breakdown product to form2-nitro-5-thiobenzoate or TNB²⁻, which has an absorbance at 412 nm. Inmalathion degradation assays, a similar trend could be detected at bothpH 7.4 and pH 4.8. OPT is less adept at cleaving thiol groups, andtherefore enzymatic degradation of malathion is relatively slow.Therefore, free OPT displayed a much lower activity of 17.1% at pH 7.4and 0.6% at pH 4.8 (FIG. 3E). However, OPT-PIMs could still maintainhigh activity of 82.2% at pH 4.8. This indicates that the benefits ofthe microparticle design are more pronounced when degrading OPs becauseOPT can degrade relatively slowly. The superior catalytic performance ofPIMs in pH 4.8 demonstrates the importance of utilizing a biomaterialelement to protect enzyme catalysts in oral consumption.

Further experiments were aimed at better understanding the stability ofthe detoxifying system under significant thermal stress, as anytreatment could experience high summer temperatures when administered tobees. OPT has been found to denature at temperatures exceeding 45° C.Thus, experiments were conducted to determine whether microparticleencapsulation offers any protection from thermal denaturation. Thecapacity for the microparticle design to withstand elevated temperatureswas tested by measuring paraoxon breakdown following enzyme incubationat temperatures ranging from 30 to 60° C. The relative enzymaticactivity was obtained by normalizing the absorbances of both free OPTand OPT-PIMs to that of OPT-PIMs incubated under 30° C. As shown in FIG.3F, free OPT displayed half the bioactivity of OPT-PIMs under 30° C. Theenzymatic activity of free OPT dramatically dropped to 36.5% afterincubation at 50° C., whereas the activity of OPT-PIMs remained at 66.3%at the same temperature. Further, increases in temperature>60° C.resulted in little catalytic activity (5.0%) of the free enzyme, whichis much lower than that of OPT-PIMs (17.7%). It was herein found thatOPT maintained greater bioactivity when encapsulated in PIMs relative tothe case of free OPT as temperatures increased. The foregoing result isimportant for the potential application of the present design, as it hasbeen shown capable of being administered at elevated temperatures.

To gauge the time taken for treatment to lose functionality, bioactivityof each group treatment relative to OPT-PIMs was measured over time whenkept at room temperature (25° C.). Microparticle activity maintainedaround 60% of original activity after 7 days and 49.5% after 14 days,whereas free enzyme activity reduced to ˜30% and <10%, respectively.OPT-PIMs stored at 4° C. maintained almost 100% activity after 14 d(FIG. 3G). The microparticle's capacity for long-term bioactivity andprotein sequestration indicates a practical shelf life of the design.Other enzyme-engineering efforts, such as genetic engineering of thecatalytic and binding pockets, introducing additional chemicalfunctionalities, such as disulfide bridges or fluorine moieties, orincorporation of additives such as sugars, polyols, detergents, polymersand amino acids, may improve the catalytic efficiency of OPT and furtherenhance its storage stability.

In vivo characterizations of PIMs. B. impatiens were used for in vivoexperimentation because colonies can be maintained indoors during thewinter in a practical and accessible box. Bumblebees are known todisplay a susceptibility to OPs comparable to A. mellifera. Tounderstand the retention performance of PIMs once consumed, bumblebeeswere fed microparticles loaded with FITC-labelled HSA (FITC-HSA-PIMs)and free FITC-HSA for 30 min, before extracting digestive tracts over a12 hour period for fluorescent microscopy analysis (FIGS. 4A and 4B).FITC displayed PIMs successfully in the crop stomach and ventriculussections of the GI tract for samples collected at 0, 1, 4 and 12 hours.During the first hour of digestion, microparticles were distributedacross both the crop stomach and ventriculus. By 1 hour, the majority ofmicroparticles had travelled out of the crop stomach, before clearanceinto the ventriculus, suggesting proventricular filtering of PIMs (FIG.4A). By 12 hours, no PIMs were observable in the crop stomach,comparable to background autofluorescence of untreated bee, but asignificant number of PIMs were still detected in the posterior sectionof the GI tract. In case of free FITC-HSA, most of the fluorescence waslocated in the ventriculus at 4 hours, while a large amount was observedin crop stomach at 1 hour (FIG. 4B). The data suggest that PIMs aredigested akin to pollen grains, thus increasing the number ofmicroparticles drawn into the ventriculus alongside pollen. Thismaximizes PIM function in detoxifying pollen as it is digested. This issignificant because OPs are often found in high quantities in pollen,which may be held in the posterior section of the ventriculus fordigestion for up to 12 h or more. The degree of fluorescence was notable to be quantified because FITC fluorescence is pH dependent; thepresence of microparticles would have altered stomach pH to the pointwhere fluorescence readings would have been inaccurate. However, theseimages qualitatively suggest that the PIM design improved retention andprovided protection from the denaturation of loaded proteins.

Efficacy and survival studies. The characterization of OPT-PIM efficacywas further validated via quantification of AChE activity when mixedwith the above described treatment and paraoxon. As AChE is inhibited byOPs such as paraoxon, high AChE activity would indicate effectivedetoxification through the above described treatment. Acetylthiocholinecleavage via AChE can be used to quantify AChE activity, as thethiocholine product reacts with DTNB to form TNB²⁻, which has anabsorbance at 412 nm (FIG. 5A). Homogenized honeybee cells were able tomaintain 91.5% of AChE activity when treated with 0.5 mM paraoxon andOPT-PIMs, relative to the positive control (homogenized honeybee cellswithout any paraoxon treatment). This was a stark improvement in AChEfunctionality relative to the negative control (homogenized honeybeecells treated with paraoxon but no free OPT or OPT-PIMs treatment),which resulted in a relative activity reduction of ˜72%. Samples treatedwith free OPT retained 18.8% less activity than that of samples treatedwith OPT-PIMs (FIG. 5B).

Groups of 50 bumblebees were treated with paraoxon ormalathion-contaminated pollen balls and OPT-sucrose treatments todetermine the efficacy of treatments in reducing mortality under OPexposure (FIG. 5C). Paraoxon and malathion present oral LD₅₀s forhoneybees at 0.0175 and 0.38 μg per bee, respectively. This data set abenchmark for OP doses for administration and subsequent detoxificationto demonstrate OPT-PIM efficacy. Bumblebees consume approximately 40.5μg pollen per day depending on body mass. Based on this figure, pollenballs were initially formed containing 0.432 μg g⁻¹ paraoxon and 9.383μg g⁻¹ malathion to feed without enzyme treatment as a negative control.It was herein found that these pollen balls caused no healthdeterioration after one week. Subsequently, through trial and error, thecontamination was significantly increased to concentrations that causedsignificant mortality. Pollen balls were tested containing 50 μg g⁻¹paraoxon over 12 hours to measure the OPT-PIM impact against acuteexposure. In this trial, OPT-PIMs at 500 μg ml⁻¹ of OPT were able tomaintain a 70% survival rate, whereas free enzyme- and sucrose-treatedgroups sustained 62.5 and 72.5% fatalities, respectively (FIG. 5D).Although OPT-PIMs largely detoxified acute exposure, the catalyticefficiency was not able to fully mitigate mortality.

A moderate level of toxicity was tested using 15 μg g⁻¹ paraoxon and 750μg g⁻¹ malathion-contaminated pollen balls against 50 μg ml⁻¹ OPTtreatments. Free OPT and no treatment resulted in 100% mortality after 5and 4 days, respectively, following paraoxon toxicity. OPT-PIMs wereable to maintain a slower incidence of mortality relative to othertreatments. After 10 days, 38% of the group sample had survived. Groupscontaining non-contaminated pollen balls and either pure sucrose orPIM-sucrose maintained 100% and 96% survival, respectively. A minorlevel of mortality is generally typical in any sample after 10 days(FIG. 5E). In malathion contaminated samples, OPT-PIM at 800 μg ml⁻¹ ofOPT was able to maintain 100% survival for the duration of observation,a lower concentration of 500 μg ml⁻¹ maintained above 80% survival over10 days. Free OPT and sucrose-treated groups presented 100% mortalityafter 5 and 4 days, respectively, analogous to the paraoxon trial (FIG.5F). Without being bound by theory, the poor performance of free,unprotected OPT may be in part driven by its higher denaturation in theacidic conditions of the digestive tract. Pollen grains are known torelease their internal contents as they progress along the midgut. Itmay be assumed that any OPs absorbed into pollen grains duringincubation are also made available at this stage of digestion. Thismeans that for the effective detoxification of contaminated pollen, OPTmust retain bioactivity until it makes passage into the ventriculus. Inaddition, it is critical that a high concentration of OPT is drawn intothe midgut to intercept paraoxon or malathion as pollen is digested.Both of these conditions have been facilitated via OPT encapsulation inPIMs.

CONCLUSIONS

Experimentation has shown that PIMs are able to enhance the bioactivityof OPT. OPT-PIMs outperform free OPT when tested under unfavorableconditions of temperature, storage and pH. The microparticle designdescribed herein rendered OPT suitable for use in pollinator OPintoxication, as it bestows functionality in gastric acidity and canmaintain performance for longer durations under elevated thermal stress.The microparticle design has also improved the functionality of OPTduring digestion by considering the bee's digestive system.Microparticles are extracted into the midgut and retained for a longduration. The aforementioned advantages ultimately result in a lowerrate of mortality when treated with OPT-PIM relative to free OPT. Thebenefits are most appreciable when degrading OPs, which are typicallyhydrolyzed at a lower relative rate, as in the case of malathion. Thiswork has produced a viable product to mitigate insecticide damage topollinator colonies and revealed new ways in which research can addressthe impacts of insecticide application through improving this currentdesign, or by exploring new microparticle treatments.

While there have been shown and described what are at present consideredthe preferred embodiments of the invention, those skilled in the art maymake various changes and modifications which remain within the scope ofthe invention defined by the appended claims.

What is claimed is:
 1. A composition comprising microparticles whereineach microparticle comprises: (i) a phosphotriesterase; (ii)nanoparticles; and (iii) a surface active agent.
 2. The composition ofclaim 1, wherein the nanoparticles are inorganic nanoparticles.
 3. Thecomposition of claim 2, wherein said inorganic nanoparticles have acarbonate composition.
 4. The composition of claim 3, wherein saidcarbonate composition is a calcium carbonate or magnesium carbonatecomposition.
 5. The composition of claim 1, wherein said nanoparticleshave an acid-scavenging composition.
 6. The composition of claim 1,wherein the nanoparticles have a size in a range of 1-500 nm.
 7. Thecomposition of claim 1, wherein the surface active agent is a polymer.8. The composition of claim 1, wherein the surface active agent isgelatin.
 9. The composition of claim 1, wherein the phosphotriesteraseand the surface active agent are dispersed throughout eachmicroparticle.
 10. The composition of claim 1, wherein saidphosphotriesterase is dissolved or suspended in an aqueous medium withineach microparticle.
 11. The composition of claim 10, wherein saidaqueous medium has an alkaline pH.
 12. The composition of claim 1,wherein said microparticles have a size in a range of 0.1-100 microns.13. The composition of claim 1, wherein said microparticles have a sizein a range of 1-100 microns.
 14. The composition of claim 1, whereinsaid microparticles possess an outer surface porosity characterized bypores having a pore size in a range of 1-500 nm.
 15. The composition ofclaim 1, wherein said microparticles possess an outer surface porositycharacterized by pores having a pore size in a range of 1-100 nm.
 16. Anaqueous suspension comprising microparticles of any one of claims 1-15suspended in an external aqueous medium.
 17. The aqueous suspension ofclaim 16, wherein said suspension contains an insect pollinatorattractant.
 18. The aqueous suspension of claim 17, wherein said insectpollinator attractant is sucrose.
 19. The aqueous suspension of claim18, wherein said sucrose is present in a concentration of 1-5 g/mL insaid external aqueous medium.
 20. A method of detoxifying insectpollinators from one or more organophosphate pesticides, the methodcomprising placing a detoxifying aqueous suspension in a locationaccessible to the insect pollinators to permit the insect pollinators toingest the detoxifying aqueous suspension, wherein said detoxifyingaqueous suspension comprises microparticles suspended in an externalaqueous medium containing an insect pollinator attractant, wherein eachmicroparticle comprises: (i) a phosphotriesterase; (ii) nanoparticles;and (iii) a surface active agent.
 21. The method of claim 20, whereinsaid organophosphate pesticide is selected from one or more of the groupconsisting of malathion, parathion, methyl parathion, chlorpyrifos,diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos,azamethiphos, azinphos-methyl, azinphos-ethyl, and terbufos.
 22. Themethod of claim 20, wherein said insect pollinators comprise the orderHymenoptera.
 23. The method of claim 22, wherein said insect pollinatorsare bees.
 24. The method of claim 20, wherein said insect pollinatorattractant is sucrose.
 25. The method of claim 24, wherein said sucroseis present in a concentration of 1-5 g/mL in said external aqueousmedium.
 26. The method of claim 20, wherein said method results in atleast 50% survival of the insect pollinators compared to insectpollinators administered said external aqueous medium without saidmicroparticles.
 27. The method of claim 20, wherein the nanoparticlesare inorganic nanoparticles.
 28. The method of claim 27, wherein saidinorganic nanoparticles have a carbonate composition.
 29. The method ofclaim 28, wherein said carbonate composition is a calcium carbonate ormagnesium carbonate composition.
 30. The method of claim 20, whereinsaid nanoparticles have an acid-scavenging composition.
 31. The methodof claim 20, wherein the surface active agent is a polymer.
 32. Themethod of claim 20, wherein the surface active agent is gelatin.
 33. Themethod of claim 20, wherein the phosphotriesterase and surface activeagent are dispersed throughout each microparticle.
 34. The method ofclaim 20, wherein said phosphotriesterase is dissolved or suspended inan aqueous medium within each microparticle.
 35. The method of claim 34,wherein said aqueous medium has an alkaline pH.
 36. The method of claim20, wherein the microparticles have a size in a range of 0.1-100microns.
 37. The method of claim 20, wherein the microparticles have asize in a range of 1-100 microns.
 38. The method of claim 20, whereinsaid microparticles possess an outer surface porosity characterized bypores having a pore size in a range of 1-500 nm.
 39. The method of claim20, wherein said microparticles possess an outer surface porositycharacterized by pores having a pore size in a range of 1-100 nm.